In breast cancer patients, the migration of cancer cells away from the primary tumor and their subsequent metastasis to distant organs is the leading cause of mortality. Metastatic cells escape the primary tumor and enter the bloodstream by developing actin-rich membrane protrusions called invadopodia that degrade the extracellular matrix (ECM) to allow invasion of surrounding tissues. The assembly of invadopodia is regulated by Rho GTPases, a family of proteins that regulates the actin cytoskeleton. However, little is known about how they are activated, the time course of their activation, or the identity of their upstream regulators and downstream effectors. Our long term goal is to characterize the Rho GTPases signaling pathways that contribute to cancer cell metastasis, in particular to cancer cell migration and invasion. The objective of this study is to characterize the molecular mechanisms that regulate RhoG in the formation of invadopodia in breast cancer. Based on our preliminary results, our central hypothesis is that RhoG functions as a negative regulator of invadopodia formation, and functions during their disassembly, in a process that involves the exchange factor SGEF and a yet to be identified RhoGAP/s. We will test our hypothesis by pursuing three specific aims: Aim 1. To identify the RhoG-specific RhoGAPs involved in invadopodia formation. We have used a novel proximity-based labeling assay coupled to mass spectrometry to identify several potential RhoG GAPs. Here, we will characterize their role in regulating RhoG activity, and their function during invadopodia formation and cell invasion. Aim 2. To characterize the role of SGEF in the regulation of RhoG during invadopodia formation. Here, we will test our working hypothesis that SGEF, a RhoG-specific GEF, is activated by Src, maintains the active pool of RhoG that prevents invadopodia formation, and needs to be inactivated for invadopodia to form. Aim 3. To characterize the spatio-temporal dynamics of RhoG activation during invadopodia formation. Based on preliminary results, our working hypothesis is that RhoG activation is tightly regulated both temporally and spatially during invadopodia formation. Here, we will use a novel RhoG FRET biosensor to determine the spatial and temporal activation pattern of RhoG during invadopodia formation in live cells, and the role of RhoGAPs and RhoGEFs in regulating its activity. The approach proposed here is innovative because it applies cutting edge techniques to examine aspects of invadopodia formation that have either not been examined before and/or have not been accessible with previously available tools. The proposed research is significant because by characterizing this pathway, it has the potential to identify novel targets that could drive the development of new therapeutics to prevent or slow down metastasis in breast cancer and other cancer types that utilize invadopodia to invade other tissues.